1. Field of the Invention
This invention relates to the use of an L-lysine .alpha.-oxidase having very high substrate-specificity with respect to L-lysine, that is, an L-lysine .alpha.-oxidase, and more particularly to the utilization of the L-lysine .alpha.-oxidase for determination of L-lysine.
2. Description of the Prior Art
L-lysine is one of the essential amino acids. The L-lysine, however, is generally contained in a lower amount in vegetable proteins and, for example, is considered as the limiting amino acid in rice, rye and corn, and as the primary limiting amino acid in wheat, sesame, oats, etc. The enrichment of L-lysine in vegetable foods and feeds has been acknowledged to be effective in improving their nutritive values and has been carried out in the world. Thus, the assay of L-lysine contained in natural foods and enriched foods is very important in view of the science of nutrition. The assay of L-lysine is also important in the nutritive conditioning of human beings and domestic animals as well as in the diagnosis of metabolic disorders. The production and demand of L-lysine have been increased in the field of food and feed industries. On the other hand, the relations between the L-lysine level in vivo and the nutritive and pathologic conditions in vivo have been clarified in the field of nutritive physiology and pathologic biochemistry. Thus, there has been a need for the establishment of a simple and sensitive method of determining L-lysine.
Examples of methods for determination of L-lysine which have hitherto been developed are chromatographic methods such as paper chromatography, thin-layer chromatography, ion-exchange chromatography, and gas-liquid chomatography specific chemical methods, microbial methods, and enzymic methods. The conventional enzymic methods include (1) determination of carbon dioxide generated from L-lysine contained in a sample by the action of L-lysine decarboxylase (cf. Methods of Biochemical Analysis, Vol. IV, pp. 285-306, 1957, Interscience Pushlishers Inc.) (2) determination of the amount of oxidized NADH when an L-lysine-containing sample is incubated with .alpha.-ketoglutaric acid and saccharopin dehydrogenase (cf. Analytical Biochemistry, Vol. 49, p.p. 225-231, 1972), and (3) determination of .DELTA..sup.1 -piperideine-6-carboxylic acid which is formed by incubating L-lysine and .alpha.-ketoglutaric acid in the presence of L-lysine: .alpha.-ketoglutaric acid .epsilon.-aminotransferase (cf. Analytical Biochemistry, Vol. 87, p.p. 283-289, 1978).
Hitherto there have been reports on the presence of L-amino acid oxidases in microorganisms, snake venom, the rat kidney, the fowl liver, and invertebrates (Arch Biochem. Biophys. Vol 146, p.p. 54-63, 1971; Journal of Bacteriology, Vol 121, No. 2, p.p. 656-662, Feb., 1975; and the Tanpakushitsu.Kakusan Koso, Vol. 17, No. 1, pp. 42-55, 1972). An L-amino acid oxidase having very high substrate-specificity to L-lysine has never been known in the art. In other words, known L-amino acid oxidases exhibit only very low enzyme activities to L-lysine except that an L-amino acid oxidase preparation derived from the turkey liver exhibits a high activity to L-lysine. However, the turkey liver enzyme also effectively oxidizes several amino acids other than L-lysine, such as L-arginine, L-hietidine and L-ornithine, at the rate equivalent to or greater than the rate of oxidizing L-lysine. Thus, the oxidase preparation can not be considered to be an enzyme having especially high substrate-specificity to L-lysine.